Methods for determination of previous ethanol consumption

Abstract

An improved method for assessing previous ethanol exposure comprises the steps of obtaining a sample from the body of a subject; quantitatively determining the level of one or several bio-precursors of phosphatidylethanol (PEth) (Formula I) or ethyl-sphingomyelin and the level of the corresponding one or several PEth (Formula II) or ethyl-sphingomyelin homologues in the sample; and obtaining a ratio between the level of one or several bio-precursors of PEth or ethyl-sphingomylein and the level of the corresponding one or several PEth- or ethyl-sphingomylein homologues. The ratio is indicative of previous ethanol consumption of the subject. Furthermore, a method for assessing previous absolute ethanol consumption can be accomplished by correlating the ratios of PEth precursor and PEth homologue levels in subjects to ratios obtained from reference subjects with known previous ethanol consumption.

The invention claimed is: 
     
       1. A method for assessment of previous ethanol exposure in a subject comprising the steps of:
 (i) obtaining a sample from the body of the subject; 
 (ii) removing one or both of the carboxylic acid residues from compounds of Formula II present in said sample by selective hydrolysis, 
 
       
         
           
           
               
               
           
         
         (iii) quantitatively determining a level of reaction product from step (ii); and 
         (iv) using said level to assess previous ethanol exposure in the subject; 
         wherein 
         the subject is a human or animal; 
         said selective hydrolysis is performed by a hydrolase selected from the group consisting of pancreatic triacylglycerol lipases, lipoprotein lipases, lingual lipase, monoacylglycerol lipases, diacylglycerol lipases, hepatic lipases, triglyceride lipases; and 
         R 1  and R 2  are independently selected from straight or branched C 9-39  alkyl, C 9-39  alkenyl, C 9-39  alkdienyl and C 9-39  alktrienyl. 
       
     
     
       2. The method according to  claim 1 , further comprising the steps of removing carboxylic acid residues covalently bound as esters in one or several bio-precursors of phosphatidylethanol (PEth) present in said sample by selective hydrolysis to yield corresponding alcohol or alcohols;
 quantitatively determining a level of said corresponding alcohol or alcohols; 
 obtaining a ratio between said level of said corresponding alcohol or alcohols and said level 
 using said ratio between said level of said corresponding alcohol or alcohols and said level of said reaction product from step (ii) in  claim 1  to assess previous ethanol exposure in the subject. 
 
     
     
       3. The method according to  claim 2 , wherein said bio-precursors of PEth are selected from the group consisting of phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin and phosphatidyl choline. 
     
     
       4. A method for assessment of previous absolute ethanol consumption in a subject comprising the steps of:
 (i) obtaining a first measure of previous ethanol exposure in the subject according to  claim 1 , said first measure being the level of said reaction product from step (ii); 
 (ii) obtaining a second measure of previous ethanol exposure in at least one reference according to  claim 1 , said second measure being said level of the reaction product from step (ii); and 
 (iii) comparing said first measure with said second measure to determine the previous absolute ethanol consumption in the subject; 
 wherein 
 said first measure is obtained by a method which is the same as the method by which said second measure is obtained; and 
 the at least one reference is a human or animal which has been exposed to a known amount of ethanol prior to obtaining a sample used for obtaining said second measure. 
 
     
     
       5. A method for assessment of previous ethanol exposure in a subject comprising the steps of:
 (i) obtaining a sample from the body of the subject; 
 (ii) removing carboxylic acid residues of compounds of Formula II present in said sample to yield a compound of Formula IIc by selective transesterification 
 
       
         
           
           
               
               
           
         
         (iii) quantitatively determining a level of said compound of Formula IIc; and 
         (iv) using said level of said compound of Formula IIc to assess previous ethanol exposure in the subject; 
         wherein 
         the subject is a human or animal; 
         R 1  and R 2  are independently selected from straight or branched C 9-39  alkyl, C 9-39  alkenyl, C 9-39  alkdienyl and C 9-39  alktrienyl; and 
         R 4  is a saturated or unsaturated straight carbon chain, or a carboxylic acid residue of a synthetically produced carboxylic acid. 
       
     
     
       6. The method according to  claim 5 , wherein at least one of R1 and R2 is different from R4. 
     
     
       7. The method according to  claim 5 , further comprising the steps of
 exchanging the carboxylic acid residues covalently bound as esters in one or several bio-precursors of PEth present in said sample by selective transesterification to yield corresponding transesterified ester or esters; 
 quantitatively determining a level of said corresponding transesterified ester or esters; 
 obtaining a ratio between said level of said corresponding transesterified ester or esters and said level of compound of Formula IIc; and 
 using said ration between said level of said corresponding transesterified ester or esters and said level of compound of Formula IIc to assess previous ethanol exposure in the subject. 
 
     
     
       8. The method according to  claim 7 , wherein said bio-precursors of PEth are selected from the group consisting of phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin and phosphatidyl choline. 
     
     
       9. The method according to  claim 5 , further comprising the steps of
 removing carboxylic acid residues from compounds of Formula I present in said sample to yield a compound of Formula Ic by selective transesterification; 
 
       
         
           
           
               
               
           
         
         quantitatively determining the level of said compound of Formula Ic; 
         obtaining a ratio between the level of said compound of Formula Ic and said compound of Formula IIc; and 
         using said ratio between the level of said compound of Formula Ic and said compound of Formula IIc to assess previous ethanol exposure in the subject; 
         wherein 
         said compound of Formula I is a bio-precursors of PEth; 
         R 1  and R 2  are independently selected from straight or branched C 9-39  alkyl, C 9-39  alkenyl, C 9-39  alkdienyl and C 9-39  alktrienyl; 
         R4 is a saturated or unsaturated straight carbon chain, or the carboxylic acid residue of a synthetically produced carboxylic acid; and 
         R 3  is selected from 
       
       
         
           
           
               
               
           
         
       
     
     
       10. The method according to  claim 9 , wherein at least one of R1 and R2 is different from R4. 
     
     
       11. The method according to  claim 9 , wherein all substituents R4 of compounds of Formula Ic are the same as all substituents R4 of compounds of Formula IIc. 
     
     
       12. A method according to  claim 9 , wherein R4 of compounds of formula Ic or IIc comprise one or several UV-absorbing groups selected from 6- or 5-membered aromatic rings or condensed aromatic systems. 
     
     
       13. A method according to  claim 5 , wherein R4 of compounds of formula Ic or IIc comprise one or several UV-absorbing groups selected from 6- or 5-membered aromatic rings or condensed aromatic systems. 
     
     
       14. A method for assessment of previous absolute ethanol consumption in a subject comprising the steps of:
 (i) obtaining a first measure of previous ethanol exposure in the subject according to  claim 5  said first measure being said level of said compound of Formula IIc; 
 (ii) obtaining a second measure of previous ethanol exposure in at least one reference according to  claim 5 , said second measure being said level of said compound of Formula IIc; and 
 (iii) comparing said first measure with said second measure to determine the previous absolute ethanol consumption in the subject; 
 wherein 
 said first measure is obtained by a method which is the same as the method by which said second measure is obtained; and 
 the at least one reference is a human or animal which has been exposed to a known amount of ethanol prior to obtaining a sample used for obtaining said second measure. 
 
     
     
       15. A method for assessment of previous ethanol exposure in a subject comprising the steps of:
 (i) obtaining a sample from the body of the subject; 
 (ii) quantitatively determining a level of one or several bio-precursors of PEth and a level of corresponding one or several PEth-homologues in said sample; 
 (iii) obtaining a ratio between said level of one or several bio-precursors of PEth and said level of corresponding one or several PEth-homologues; and 
 (iv) using said ratio between said level of one or several bio-precursors of PEth and said level of the corresponding one or several PEth-homologues to assess previous ethanol exposure in the subject; 
 wherein 
 the subject is a human or animal. 
 
     
     
       16. The method according to  claim 15 , wherein said one or several PEth-homologues are independently selected from the group consisting of PEth 16:0/18:1, PEth 16:0/18:2, PEth 16:0/20:4, PEth 18:0/18:1, PEth 18:1/18:1 and PEth 18:0/18:2, or any one of the corresponding regioisomers in which the carboxylic acid residues have switched places on the glycerol backbone. 
     
     
       17. The method according to  claim 15 , wherein said one or several bio-precursors of PEth are independently selected from the group consisting of phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin and phosphatidyl choline. 
     
     
       18. The method according to  claim 15 , wherein said ratio is
 a ratio between the sum of concentrations or the sum of amounts of at least two bio-precursors of PEth in said sample and the sum of concentrations or the sum of amounts, respectively, of at least two PEth-homologues in the same sample; or 
 a ratio between the sum of concentrations or the sum of amounts of at least two PEth-homologues in said sample and the sum of concentrations or the sum of amounts, respectively, of at least two bio-precursors of PEth in the same sample; 
 each one of said at least two PEth-homologues being a compound formed in-vivo from one of said at least two bio-precursors of PEth in the presence of ethanol. 
 
     
     
       19. A method for assessment of previous absolute ethanol consumption in a subject comprising the steps of:
 (i) obtaining a first measure of previous ethanol exposure in the subject according to  claim 15 , said first measure being said ratio obtained in step (iii) of  claim 15 ; 
 (ii) obtaining a second measure of previous ethanol exposure in at least one reference according to  claim 15 , said second measure being said ratio obtained in step (iii) of  claim 15 ; and 
 (iii) comparing said first measure with said second measure to determine the previous absolute ethanol consumption in the subject; 
 wherein 
 said first measure is obtained by a method which is the same as the method by which said second measure is obtained; and 
 the at least one reference is a human or animal which has been exposed to a known amount of ethanol prior to obtaining a sample used for obtaining said second measure. 
 
     
     
       20. A method for assessment of previous ethanol exposure in a subject comprising the steps of:
 (i) obtaining a sample from the body of the subject; 
 (ii) quantitatively determining a level of one or several bio-precursors of ethyl-sphingomyelin and a level of corresponding one or several ethyl-sphingomyelin homologues in said sample; 
 (iii) obtaining a ratio between said level of one or several bio-precursors of ethyl-sphingomyelin and said level of the corresponding one or several ethyl-sphingomyelin homologues; and 
 (iv) using said ratio between said level of one or several bio-precursors of ethyl-sphingomyelin and said level of the corresponding one or several ethyl-sphingomyelin homologues to assess previous ethanol exposure in the subject; 
 wherein 
 the subject is a human or animal. 
 
     
     
       21. A method for assessment of previous ethanol exposure in a subject comprising the steps of:
 (i) obtaining a blood sample of the subject; 
 (ii) quantitatively determining a level of one or several PEth-homologues, or a level of one or several ethyl-sphingomyelin homologues, in said blood sample, and determining a level of a measurable parameter representing an amount of cellular material in said blood sample; 
 (iii) obtaining a ratio between said level of one or several PEth-homologues or said level of one or several ethyl-sphingomyelin homologues, and said level of said measurable parameter; and 
 (iv) using said ratio between said level of one or several PEth-homologues or one or several ethyl-sphingomyelin homologues, and said level of said measurable parameter to assess previous ethanol exposure in the subject; 
 wherein 
 the subject is a human being or animal; and 
 said measurable parameter is haemoglobin or the erythrocyte volume fraction. 
 
     
     
       22. A method for assessment of the history of previous ethanol exposure in a subject comprising the steps of:
 (i) obtaining a sample from the body of the subject; 
 (ii) quantitatively determining a level of one or several bio-precursors of PEth, or a level of one or several bio-precursors of ethyl-sphingomyelin, and a level of corresponding one or several first bio-markers of ethanol being PEth or a level of one or several ethyl-sphingomyelin homologues, respectively, and quantitatively determining a level of a second bio-marker of ethanol; 
 (iii) comparing said level of one or several bio-precursors of PEth or said level of one or several bio-precursors of ethyl-sphingomyelin, said level of the corresponding one or several first bio-markers of ethanol, and said level of said second bio-marker of ethanol to determine the history of previous absolute ethanol consumption in the subject; 
 wherein 
 the subject is a human or animal.